Dephospho-CoA Kinase Provides a Rapid and Sensitive Radiochemical Assay for Coenzyme A and Its Thioesters

Anal Biochem. 2007 Sep 1;368(1):17-23. doi: 10.1016/j.ab.2007.05.031. Epub 2007 Jun 7.

Abstract

A new approach to determine in vivo pools of coenzyme A (CoA) and short chain acyl-CoA thioesters is reported. The metabolites released by extraction with trichloroacetic acid are recovered and quantitatively dephosphorylated by treatment with shrimp alkaline phosphatase. Following phosphatase removal, the dephosphorylated CoA metabolites are quantitatively rephosphorylated by treatment with [gamma-33P]ATP plus a dephospho-CoA kinase. The resulting radioactive CoA metabolites are then separated by reverse-phase high-performance liquid chromatography and quantitated by scintillation counting. Due to the enzymatic radiophosphorylation, the assay is specific for CoA and its short chain thioesters and is sensitive to sub-picomole levels of these compounds.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Acyl Coenzyme A / analysis*
  • Biological Assay
  • Chromatography, High Pressure Liquid*
  • Esters / analysis
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism*
  • Scintillation Counting

Substances

  • Acyl Coenzyme A
  • Esters
  • Phosphotransferases (Alcohol Group Acceptor)
  • dephospho-CoA kinase